PROJECT SUMMARY The goal of the Phase II grant is to expand the Phase I demonstration of improvements to protein crystallization achieved using engineered nucleation surfaces to other high impact targets that include proteins involved in antibiotic resistance and membrane proteins.? ?The role of crystallography in establishing the structure of biological macromolecules plays a key role in understanding their function, and Public Health benefits will derive from more efficient targeted drug development. Crystallization of proteins remains a challenge, and current approaches are experimentally intensive and have >75% failure rates, which creates a ?crystallization bottleneck? that is the rate limiting step in structural biology. Phase I Aims were accomplished using surface energy modifications giving an average reduction in crystallization onset times of ?31% with a 2-4 fold increase in the number of crystals/experiment, of which 74% formed on the engineered nucleation features for bovine pancreatic trypsin, thaumatin, lysozyme, and ???-lactoglobulin? (n = 15-26 each). Phase II results with the first challenge protein PA01 (also known as NP_254171.1, provided by the Center for Structural Genomics of Infectious Diseases at Argonne) are even more remarkable: the engineered nucleation features improved crystallization hits by 8.4-fold and produced a 6-fold increase in crystals in 87% less time. ?DeNovX?s products will be HTS plates for crystallization, and a slot-in strategy will foster technology adoption by fitting into existing workflows and equipment. The innovative aspects of the patented technologies are surface modifications that give tunable surface energies that enhance crystal nucleation. Our hypothesis is that success outcomes for difficult to crystallize proteins can be improved using surface energy modifications to improve crystal nucleation. Specific Aim 1 - Expand the ongoing probe of crystallization function to protein targets involved in the emerging threats from antibiotic resistance (e.g., ???-lactamases, etc.). Specific Aim 2 - Initiate systematic investigations that advance the mechanistic understanding of how different surface energy modifications improve crystal nucleation. ?This understanding will pave the way for rational tailoring of the surface energy modifications for use with other soluble and ?membrane proteins. Specific Aim 3 - Expand end user research and prototyping and initiate ???-site testing making HTS formats with surface energy nucleation enhancements available for use in membrane protein facilities. This equipment request seeks to fund acquisition of a dynamic light scattering (DLS) plate reader that will permit the investigators to quantitatively probe protein aggregation in samples to be crystallized. Protein aggregation can inhibit or enhance crystal nucleation, and a better understanding of protein aggregation size characteristics as a function of solution conditions and surface energy modifications will better equip the investigators to improve crystallization outcomes for high impact proteins and to diagnose the issues for those proteins that have failed prior screening attempts.